Maintenance of genomic stability is critical for the well-being of organisms. To maintain genomic stability, cells have developed a network of signaling pathways, collectively known as the DNA damage response (DDR) pathway, to sense and repair DNA damage. In response to DNA damage, many factors involved in the DDR, such as MDC1, 53BP1, and BRCA1 get recruited to the sites of DNA damage to facilitate cell cycle checkpoint activation and DNA repair. We and others have studied the molecular mechanisms by which BRCA1, 53BP1 and NBS1 are recruited to the sites of DNA damage through MDC1. More recently, we found that disassembly of DDR factors including MDC1 is also important for proper DDR. These observations led us to hypothesize that dynamic assembly and disassembly of MDC1/RNF8/BRCA1/53BP1 is critical for the maintenance of genomic stability. In our preliminary data, we found that several E3 ubiquitin ligases play an important role in this process. We found that RNF4 and PHRF1 negatively regulate the accumulation of MDC1 and RNF8 respectively. We plan to further test our hypothesis with the following Specific Aims: Aim 1. Study the mechanism of cell cycle specific removal of MDC1by RNF4. Aim 2. Study the regulation of the MDC1-RNF8 pathway by PHRF1. Aim 3. Study the role of PHRF1 in tumorigenesis using animal models. The proposed studies are an underexplored area in the field DNA damage response. We believe that successful completion of these studies will significantly advance our understating of the temporal and spatial regulation of DDR. In addition, RNF4 and PHRF1 are all found to be misregulated in human cancers. Our studies could also reveal important insight into molecular oncogenesis and cancer therapy.